Inclusion of juvenile stages improves diversity assessment and adds to our understanding of mite ecology – A case study from mires in Norway

Abstract Arachnid orders, Mesostigmata, Trombidiformes, and Sarcoptiformes, commonly known as ‘mites’, are abundant in mires, both as adults and as juveniles. However, due to the challenges of identification, the juvenile forms are often excluded from analyses. This is the first study in mires that included all three mite orders identified to the species level, including juvenile instars. We aimed to compare how diversity and the response to ecological variables differed if only the adults (ad) vs. the total number of specimens (ad+juv) are considered. Samples of 20 Sphagnum species (five subgenera) were collected and mites were extracted using Berlese funnels. Overall, nearly 60,000 mites were analyzed; of these Mesostigmata made up 1.87% of the total, Trombidiformes −0.27%, and Sarcoptiformes −97.86%. The study revealed 154 species (33 Mesostigmata, 24 Trombidiformes, and 97 Sarcoptiformes), the highest diversity of mites ever reported from mires. The inclusion of juveniles increased observed species richness by 6%, with 10 species (one Mesostigmata, six Trombidiformes, and three Sarcoptiformes) represented only by juvenile forms. Seventeen species are new to Norway (four Mesostigmata, one Sarcoptiformes, and 12 Trombidiformes, including five undescribed species of Stigmaeidae and Cunaxidae). Four of these were represented in the samples only by juveniles. Including the juveniles explained a greater amount of the variability of Trombidiformes (explanatory variables account for 23.60% for ad, and 73.74% for ad+juv) and Mesostigmata (29.23% − ad, 52.91% − ad+juv), but had less of an impact for Sarcoptiformes (38.48% − ad, 39.26% − ad+juv). Locality, Sphagnum subgenus and species, wetness, and trophic state significantly affected the mite communities and should be taken into consideration when studying mires. Since juvenile stages contribute significantly to mite diversity in mires, they should also be included in mite studies in other habitats.


| INTRODUC TI ON
Peatlands, including mires (i.e., living peatlands that can accumulate peat) host highly specialized and unique flora and fauna, due to their nutrient-poor, acidic, and water-saturated conditions. As such, they make a significant contribution to global biodiversity.
They store twice as much carbon as all the world's forests, which is of paramount importance in relation to climate change, in particular temperature increase and changes in precipitation (Parish et al., 2008). They are also important water regulators, as they, like huge sponges, accumulate water during wet seasons and release it during dry seasons . In Norway, peatlands cover 13.8% of the country and are the best preserved in Europe . About 85% of them are still able to accumulate peat, while the average value in Europe is 50%, and in many countries, it is even lower (e.g., Finland 40%, Ireland 18%, Poland 16%, and Germany 2%) . Classification of mires varies in European countries according to each country's traditions . In Norway, the common practice is to classify mires according to hydromorphological patterns (mire massif types, Moen in Joosten et al., 2017 p. 540) and vegetation types (Moen in Joosten et al., 2017 p. 18), and without formal phytosociological classification. The main vegetational classification divides mires into ombrotrophic mires (bogs) fed by precipitation nutrients only, and minerotrophic mires (fens) fed by an additional supply of mineral soil nutrients from the mineral soil surroundings. The classification pattern found on local mire sites is primarily based on the variation in vegetation along two eco-gradients, (1) the 'poor-rich' gradient, reflecting increased nutrient conditions of the mire habitat (trophic gradient) and (2) the 'dry-wet' gradient, reflecting increased wetness conditions of the mire habitat (wetness gradient).
Mires are considered species-poor habitats (Rydin & Jeglum, 2006), but some groups of organisms, in particular Sphagnum mosses (Laine et al., 2018) and mites (Vilkamaa, 1981), are abundant and highly diverse within them. Norwegian mires have the most species-rich peat moss flora in Europe, with 54 named species, that is, 90% of all known European species (Laine et al., 2018). They also host the highest diversity of the oribatid mites (a group within Sarcoptiformes) ever found in this type of habitat, largely explained by the large Sphagnum diversity . In limited sampling in Norwegian mires, up to 95 species of Oribatida were found (i.e., 30% of known species in Norway), which is comparable to the diversity of this group in broadleaf forests (Seniczak, Niedbała, Iturrondobeitia, et al., 2021;Seniczak, Seniczak, Graczyk, et al., 2021;Seniczak, Seniczak, Starý, et al., 2021). These included 18 new records for Norway and two species new to science . The latter demonstrates that the potential of mires for the protection of species diversity associated with this specific habitat is still underestimated.

T A X O N O M Y C L A S S I F I C A T I O N
Biodiversity ecology F I G U R E 1 Nanhermannia coronata (Sarcoptiformes, Oribatida), adult; the most abundant mite species collected in this study. Solhøy, 1979). Some studies, however, indicated the importance of including juvenile instars. For example, in a study on the effect of warming on oribatid mites, significant differences in the ratio of immature stages to adults were observed in different types of fens . In Norwegian mires, juveniles constituted nearly 40% of oribatids; in some species, they highly dominated the age structure (up to 80% in Nothrus spp.), and two oribatid species were represented only by juveniles (Seniczak, Seniczak, Iturrondobeitia, et al., 2020). Similarly, juvenile forms can be important for answering ecological questions in other ecosystems, such as forests (Proctor et al., 2002). For example, the ratio of adults to juveniles can be a useful measure of forest habitat disturbance (Maaroufi et al., 2022).
The postembryonic development of mites includes several instars. The full development that occurs in Sarcoptiformes and many Trombidiformes includes a hexapod (i.e., three-legged) larva, three octopod (four-legged) nymphal stages (protonymph, deutonymph, and tritonymph) and adult. In some groups (e.g., Mesostigmata and some Prostigmata within Trombidiformes), the number of instars is reduced ).
Identification of juvenile stages is laborious and often more challenging compared to adults, due to their smaller body size, more delicate structures, and above all, because for many mite species these forms remain unknown (Solhøy, 1979). For example, among Oribatida, the juveniles of only about 8% of the known oribatid mite species and 30% of genera have been described (Norton & Ermilov, 2014). For mesostigmatid mites, it is difficult to assess the percentage of species with full ontogeny known; however, in most taxa, only adults are described. In some species groups, there are also taxa known only as juveniles; for example, in the Trichouropoda ovalis group, Hirschmann and Wiśniewski (1986) described 45 species, and about half of them (53%) are only known as deutonymphs. Among Trombidiformes the situation varies. In terrestrial Parasitengonina (Calyptostomatoidea, Erythraeoidea, and Trombidioidea), the proportions are reversed due to greater interest in parasitic larvae, particularly chiggers (Mąkol & Wohltmann, 2013); moreover, the heteromorphism of active instars resulted in independent descriptions of species known from larvae and from active postlarval forms. Of the approximately 5000 species assigned to this group, almost 80% have only been described as juveniles, and the share has been largely influenced by vertebrate-associated chiggers (Mąkol & Wohltmann, 2013). For an array of species, only the reference to the postlarval form (without distinction between juvenile deutonymph and adult form) has been provided in the literature. Among the water mites (Hydrachnidia) of the northern hemisphere, juvenile stages are known for almost all genera, but probably for <10% of the species (Prasad & Cook, 1972;Tuzovsky, 1987Tuzovsky, , 2011Wainstein, 1976Wainstein, , 1980Zawal, 2008). Among the other Prostigmata, the state of knowledge varies considerably between taxa; however, the majority of species descriptions are based on the adult instars, with juveniles included more haphazardly, depending on the taxonomist and the specimens available.
Within Trombidiformes, Parasitengonina (including both terrestrial species and Hydrachnidia) have a very complex life cycle with only some stages being active (larvae, deutonymphs, and adults) while the protonymph and tritonymph are calyptostatic, that is, nonfeeding and non-motile forms (Grandjean, 1938). Within the active stages, the larvae have very different feeding habits from the predaceous deutonymphs and adults, being generally parasitic on arthropods or vertebrates. Due to their parasitic habits, often with flying insects as hosts, these larvae are underrepresented compared to deutonymphs and adults in samples extracted in Berlese or Tullgren funnels (e.g., Wohltmann et al., 2006). Most other Prostigmata have less complex life cycles, with immature stages resembling adults both morphologically and ecologically, and co-existing with adult populations. The most extreme change is from hexapod larva to octopod nymph, with the remaining changes pertaining to increasing complexity (e.g., increasing setal counts and increasing number of genital papillae; . In most Sarcoptiformes, the juveniles co-occur in the same microhabitat as adults (although they may occupy different ecological niches by having different food preferences). But, for example, in ptyctimous mites, the juveniles live in galleries inside dead wood or conifer needles and cannot be extracted with simple methods like Berlese or Tullgren apparatus (Hågvar, 1998;Niedbała, 1992).
In the present study, three mite orders (Mesostigmata, Trombidiformes, and Sarcoptiformes) in peatlands are investigated for the first time at the species level, including both adult and juvenile forms. The aim of this paper is to compare how species diversity (measured by species richness) and ecological patterns are affected if only the adults (ad) vs. the total number (ad+juv) are considered.
Because juveniles are found abundantly in mires, we hypothesized that including juveniles will i) result in discovery of the higher species diversity of all mite groups, and ii) improve our understanding of the effect of ecological factors on the variability of mite communities.

| Sampling and material analysis
The sampling was carried out in six mires located in the western, oceanic part of Norway (Table 1, Figure 2). The climate of the region is mild and relatively warm (the average annual temperature is 6.8°C)  (Table 1), and four were from Northern Atlantic wet heath, which is a slightly different habitat, although it often forms peat. Sphagnum mosses were identified to species classified into five subgenera: Sphagnum (four species), Rigida (two species), Cuspidata (seven species), Subsecunda (one species), and Acutifolia (six species).
We aimed to collect as many different Sphagnum species as we could find; equal numbers of samples were not collected at each mire since homogeneous samples of the desired size were not always present.
The nomenclature of Sphagnum spp. with author citations of species, as well as the subordinate classification in subgenera instead of sections, follow Laine et al. (2018).
In each of the 27 sampling plots, plant species within a 1 m × 1 m plot were identified. Based on floristic composition and relative abundance of indicator species, the samples collected in true mires were assigned to trophic state (poor, intermediate, and moderately rich) and wetness gradients: hummocks (mounds of peat), lawns (firm turf-like vegetation), and carpets (softer than lawns, including quaking mats). Samples collected in wet heath were excluded from the analyses of the effect of trophic state and wetness gradients.
Vegetation data are included in Appendix S1.
Samples were transported in plastic bags to the laboratory at the University of Bergen, stored at 4°C until the next day, and extracted using Berlese funnels for 2 weeks into 70% ethanol; the temperature above the sample was approximately 30°C, and additional ethanol was added as needed due to evaporation. All active life forms of mites that were obtained during the extraction were identified, including adults and juvenile instars, that is, larvae and nymphs.
Terrestrial Parasitengonina were cleared in KOH and mounted on microscopic slides using Faure's fluid. For identification, we followed Wohltmann et al. (2006) and Łaydanowicz and Mąkol (2010).
Adults of Hydrachnidia were dissected, slide mounted in Hoyer's medium, and identified by keys of Di Sabatino et al. (2010) and Gerecke et al. (2016). The classification of higher systematic categories follows Lindquist et al. (2009a).

| Statistical analyses
The statistical analyses were based on the abundances of adults and juveniles in each mite group. All multivariate analyses were performed using CANOCO software (Microcomputer Power, Ithaca, NY, USA; Jongman et al., 1995;Ter Braak, 1988). Response data (biological species) were log-transformed, log (x + 1) (Łomnicki, 2017) in order to down-weight rare species. Independent variables were focused only on the significant variables that explained most of the variation of mite communities in the conditional effects space.

| Factors affecting mite communities
According to CCA, when all factors were considered together, they were not significant for mite orders; however, forward selection appeared significant for some factor levels, with the results differing between the ad only and ad+juv datasets (Table 3). When ad+juv were included, more levels of factors were significant (Figures 4-6, compare a vs. b).

TA B L E 2
New species records for Norway; ad -adults, juv -juveniles; for site abbreviations see  Locality KL was also the most important factor for Sarcoptiformes (Table 3)

| D ISCUSS I ON AND CON CLUS I ON S
This is the first study that demonstrates such a high diversity of mites in mires, highlighting the importance of these ecosystems for maintaining biodiversity. It is especially significant that mires host many unique species, which cannot be found in any other habitats. As many as 154 mite species were found with 20 Sphagnum species collected, including 33 species of Mesostigmata, 24 species of Trombidiformes, and 97 of Sarcoptiformes. In comparison, in other mires, the number of species found was lower, although Sarcoptiformes (or its suborder Oribatida) were always the most diverse group. For example, in northwestern Russia, 60 species of Sarcoptiformes, 15 species of Mesostigmata, and three species of Trombidiformes were found (Philippov et al., 2021), in Ireland, 43 species of Oribatida and 14 of Mesostigmata were recorded (Wisdom et al., 2011), and in southern Germany, 52 species of Oribatida were noted (Lehmitz et al., 2020).
In the latter study, the discovered species richness of Oribatida was higher than that of vegetation and beetles, but lower compared to the richness of spiders (Lehmitz et al., 2020). Lower numbers of species found in the above-mentioned studies can certainly be related to different sampling efforts or extraction efficiency (Minor et al., 2016), but the fact that only adults were identified to the species level is also of importance.
This study showed clearly that including juveniles is important for discovering mite diversity in mires, thus supporting our first hypothesis. Ten species (one Mesostigmata, six Trombidiformes, and three Sarcoptiformes) were represented only by juveniles.
Furthermore, among 17 new species records for Norway, four species (all Trombidiformes) were represented in the samples only by the juvenile instars.
The high diversity of mites in Norwegian mires can also result from the fact that these mires are well preserved  and are rich in diverse Sphagnum mosses F I G U R E 3 Average abundance (A, in 1000 ind./m 2 ) (bars) of mites with standard deviation (whiskers), and average number of species (above bars) in mires in western Norway; the proportion of adults (ad) and juveniles (juv) is marked with bars of different shades; detailed information about the sampling locations is in Table 1.   (Flatberg, 2013). Peatlands dominated by Sphagnum host significantly richer mite fauna than, for example, those dominated by Carex . Habitat complexity is undoubtedly one of the most important factors in structuring biotic assemblages (Kovalenko et al., 2012). Different peat mosses vary considerably in their photosynthetic capacity (i.e., the maximum rate at which leaves can fix carbon during photosynthesis), productivity (Breeuwer et al., 2008), decomposition rate (Limpens & Berendse, 2003), peat accumulation, litter quality (Bengtsson et al., 2016), desiccation tolerance, and recovery ability (Hajek & Vicherová, 2014;Rydin, 1986Rydin, , 1993. Different Sphagnum species also differ in the structure of their microbial communities (Bragina et al., 2012;Opelt et al., 2007). These differences are likely to influence the communities of other organisms inhabiting peat mosses, such as mites.
Oribatida are the most well-studied mite group in mires, with more than 400 species recorded in the Holarctic region (Mumladze et al., 2013). Peat mosses provide a variety of foods to different feeding groups of Oribatida. Some oribatid species feed directly on Sphagnum tissue, contrary to the belief that peat mosses are not eaten by any herbivores (Rydin & Jeglum, 2006), but most species feed on associated algae, fungi, bacteria, protozoa, and nematodes (Lehmitz & Maraun, 2016). Oribatid mites can live in different parts of Sphagnum: on drier apical parts; in more basal parts; or in the spaces between Sphagnum leaves. Oribatida are closely dependent on microhabitat conditions, most notably the moisture level (Lehmitz et al., 2020) or the genus of Sphagnum present (Minor et al., 2016;Seniczak, Seniczak, Iturrondobeitia, et al., 2020;Seniczak, Seniczak, Schwarzfeld, et al., 2020), but also water chemistry . Therefore, any changes in peatlands, either due to climate change or human activities that affect water level or water chemistry, have strong impacts on the oribatid communities (Lehmitz et al., 2020;Markkula 1981Markkula , 1982Seniczak et al., 2016;. Dramatic changes in oribatid communities can be observed very quickly, even between different seasons (Seniczak, Seniczak, Graczyk, et al., 2019), so Oribatida seem to be very good bioindicators for short-term changes in peatlands (Lehmitz et al., 2020;. Mesostigmata are less investigated in mires than Oribatida. They have been studied, for example, in Poland and Latvia, including both juvenile and adult forms, and although the number of species varied between the types of mires (10-35), several rare species new to these countries have been found. Mire communities include habitatspecific Mesostigmata species that makes these habitats particularly important in terms of biodiversity (Kaczmarek et al., 2006(Kaczmarek et al., , 2011Kaczmarek & Marquardt, 2007, 2008Marquardt & Kaczmarek, 2009;Salmane, 2006Salmane, , 2009Salmane & Spuņģis, 2015;Skorupski et al., 2008). The bioindicative reaction of Mesostigmata is less pronounced, that is, in contrast to Oribatida, none of the environmental variables were significantly associated with variation in the Mesostigmata communities, but the Mesostigmata results often support those of Oribatida (e.g., high dominance of aquatic species or a shift to generalists is often observed in both groups) .
Trombidiformes (except for water mites) are almost unknown from mires. Although these mites have been included in some studies, they were treated as a whole (e.g., Laiho et al., 2001;Seniczak, Seniczak, Graczyk, et al., 2019), without resolution to the species level. One of the trombidiform groups important in mires is the cohort Parasitengonina which comprises two main ecological groups: water mites (Hydrachnidia) and terrestrial Parasitengonina. In Europe, there have been a large number of studies on water mites inhabiting peatlands (e.g., Smit & van der Hammen, 1996;Stolbov et al., 2018;Więcek et al., 2012Więcek et al., , 2013aWięcek et al., , 2013b, and water mites seem to be represented in these habitats by relatively few species. For example, in Canada, where more than 500 species of water mite species are known, only about 30 were found in peatlands (Smith, 1987). In our study, we only collected three species of Hydrachnidia, but the methodological approach used (extraction of peat mosses) is not sufficient to discover the diversity of this group (Stryjecki et al., 2017;Więcek, Martin, & Gąbka, 2013).
The remaining terrestrial Trombidiformes, along with Endeostigmata (Sarcoptiformes), form the most heterogeneous group of mites and are found in a wide variety of environments.
It is, therefore, difficult to characterize these mites as a whole, regarding their ontogeny, biology, ecology, and habitat preferences.

F I G U R E 4
Results of canonical correspondence analysis (CCA) of Mesostigmata communities (represented by 20 best fitted species, represented by blue triangles, see Appendix S1 for species name abbreviations and environmental variables (see the Methods for the explanation)) in mires of western Norway, represented by red triangles. Names of species (or their abbreviations) are in Italic font. (a) Adults, total variation is 4.23084, explanatory variables account for 29.23% (adjusted explained variation is 18.62%).

F I G U R E 5
Results of canonical correspondence analysis (CCA) of Trombidiformes communities (represented by 10 best fitted species, represented by blue triangles, see Appendix S1 for abbreviations) and environmental variables (see the Methods for the explanation) in mires of western Norway, represented by red triangles. Names of species (or their abbreviations) are in Italic font. (a) Adults, total variation is 8.47626, explanatory variables account for 23.60% (adjusted explained variation is 11.84%). (b) Adults + juveniles, total variation is 5.74344, explanatory variables account for 73.74% (adjusted explained variation is 57.99%). They can be found in extremely different climatic zones and habitats, from very moist to very dry (Walter, 2009; Even though Trombidiformes were the least numerous group in the mires studied, their inclusion significantly increased our knowledge about the biodiversity of mires. Of the 17 species newly recorded for Norway, most were Trombidiformes (12). In addition, five of these species are considered new to science. These findings confirm that mires are fascinating and undiscovered habitats, and even in the relatively well-studied northern hemisphere new species of mites can be discovered Seniczak & Seniczak, 2009a.
Including the juvenile stages in the analyses was also very important in allowing us to better explain the variability of mite communities, what agreed with our second hypothesis. The variability of Mesostigmata was best explained by Sphagnum subgenus Cuspidata.
Another important factor was the Sphagnum species (S. riparium) with its characteristic species Parathyas pachystoma. This water mite is characteristic of vernal, astatic waters, and occurs in semi-aquatic habitats (quagfens, swamps) as well (Di Sabatino et al., 2010).
Locality KL was also the most important factor for Sarcoptiformes, with several aquatic Oribatida distinguishing this locality. Hydrozetes and Limnozetes encompass truly aquatic species (Schatz & Behan-Pelletier, 2008) and Pilogalumna tenuiclava is characteristic of moist mire habitats . Another important factor was Sphagnum species, that is, S. angustifolium was also characterized by aquatic Oribatida.
Regardless of the undisputed impact on the assessment of biodiversity, the inclusion of juvenile stages in the research also significantly contributes to the understanding of the biology and life cycle of species whose taxonomy and systematics are based on adult stages. This is of particular importance in the case of species with a specific phenology, for example, with the appearance of juvenile stages limited to short periods throughout the year. For the identification of juvenile stages, in the case of species whose identification is based on morphological features, molecular analysis may be extremely helpful.
In summary, contrary to the established convention that peatlands are species-poor habitats, well-preserved mires like the ones studied here hide significant diversity treasures. In exploring peatlands, it is important to include often ignored mite groups, for example, within order Trombidiformes, as well as taking into account the often-overlooked juvenile stages.

ACK N OWLED G EM ENTS
This study is dedicated to the late Professor Torstein Solhøy

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.